Cyclic GMP-AMP synthase (cGAS) is the primary sensor for cytosolic dsDNA, producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid-lineage cell types and responsible for providing innate immunity. Aberrant cytosolic dsDNA contributes to inflammatory diseases, suggesting that inhibition of cGAS may be therapeutically beneficial. We recently developed a mass- spectrometry-based high throughput screen (HTS) using mouse cGAS and identified small-molecule inhibitors that yielded the first active and specific inhibitors of cGAS in mouse cellular assays, albeit with no inhibitory activity in human cells. We now developed a faster and more cost effective HTS for identification of human cGAS inhibitors using chemiluminescence and screened 300,000 compounds. Subsequent medicinal chemistry optimization identified potent and specific inhibitors for human cGAS active in major interferon- producing cell types including primary macrophages and PBMCs. We have also solved co-crystal structures of inhibitors with human cGAS, suggesting structure- and computational-guided optimization path for the lead inhibitors. We propose to identify new hit compounds to access additional drug scaffolds through extended drug library screening to continue supporting our medicinal chemistry program. Using the most potent and specific inhibitors, we wish to characterize their inhibitory effect and pathway specificity in a more diverse range of cells. Our long-term goal is to develop therapeutics for cGAS-dysregulation associated diseases. This proposal is organized in 3 aims: (1) Identify new chemical scaffolds by HTS and apply cheminformatic analysis and medicinal chemistry hit optimization. We will use our newly established and validated HTS method for identification of human cGAS inhibitors to screen a newly available library of 100,000 compounds. New hit compounds will be prioritized for further derivatization to optimize potency and drug-like characteristics. (2) Structure/computation-guided design for optimization of new scaffolds and lead human cGAS inhibitors. We will use the insights gained from inhibitor-cGAS co-crystal structures to design, synthesize, and test derivatives to probe for the most potent, and promising drug-like cGAS inhibitor(s). (3) Develop new cellular validation assays for the identification of potent and highly selective cGAS inhibitors. We propose to evaluate and validate new and recently identified inhibitors by assessing their efficacy, selectivity, and biocompatibility using established and new cellular assays including non-myeloid cancer cell lines with chromosomal instability, primary myeloid cells, and patient-derived myeloid cells. Although initially discovered as central dsDNA sensor for antiviral activity, cGAS is also emerging as a player in obesity, neurodegenerative diseases, tumorigenesis, and cancer metastasis. Well-characterized, validated, potent, and specific cGAS inhibitors are needed in the community studying cGAS-STING biology as well as for treating cGAS-related inflammatory diseases!